This invention relates to parasexual hybridization.
In parasexual hybridization, protoplasts of higher plant cells are fused and some heterokaryons form a callus mass which under some circumstances differentiates into apparently normal, fertile, new plants. This procedure is disclosed by Carlson, P.S. et al; Proc. Natl. Acad. Sci., 69, 2292-2294 (1972).
In one technique of parasexual hybridization, somatic cells of different plants are treated in suitable osmotica containing high concentrations of commercial polysaccharidase preparations until their cell walls have become degraded and the protoplasts emerge. Such techniques have been described by Bajaj, Y.P.S. in Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, pp 467-577, Springer-Verlag (1977).
After the protoplasts emerge, they are subjected to conditions that cause some pairs to fuse and form heterokaryons. Several nonspecific chemical treatments induce homologous and heterologous fusion, even to the extent of the creation of inter-kingdom hybrids. The most popular nonspecific chemical fusogen is polyethylene glycol as described by Kao, K.N., Molec. Gen., Genet. 150, 225-230 (1977).
Some of the heterokaryons initiate cell wall deposition and cell division and may develop into normal plants. Techniques for such regeneration are discussed in Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, pp 467-577, Reinert, J., Bajaj, Y.P.S. ed. Springer-Verlag (1977). Specific selection schemes allow the isolation of interspecific hybrid callus for such regeneration.
In the prior art techniques of hybrid parasexual hybridization, several different techniques are used for the identification and selection of true binucleate heterokaryons from fused and unfused parental protoplasts, but at present, no general prior art methodologies are available that can achieve this identification and selection in a satisfactory manner.
Some of the prior art methods which are most significant are those which rely upon: (1) separation in accordance with the known different growth requirements of the callus tissue; (2) differential sensitivities of parental tissue to drugs and metabolic inhibitors; (3) microscopical examination of cytological markers during fusion and cultivation of protoplasts; (4) complementation of auxotrophic parental tissue following protoplast fusion and culture in a minimal medium; and (5) density gradient centrifugation to separate parental protoplasts of differing buoyant densities from hybrid protoplasts of intermediate density.
None of the above techniques have been entirely satisfactory. Each of them has had certain disadvantages such as: (1) it is difficult to predict the characteristic of the somatic hybrid upon which selection is to be based such as the growth requirements, differential sensitivities to drugs and metabolic inhibitors or the like; (2) suitable parental tissue is not available with characteristics that complement to form a characteristic suitable for selection such as susceptibility to drugs, susceptibility to light, weight differences, or natural differentiation characteristics to serve as markers or the like; and (3) certain of the techniques put too much strain on the cells.
Fluorescent labeling has been used in the prior art for the identification and sorting of cells. It has been used sucessfully in the sorting of animal cells by formation of antibodies to the animal cells and attachment of the label to the antibody for later attachment to cells.
The growing of higher plant cells in culture and the labelling of such cells by fluorescence marking was reported by D. W. Galbraith and J. E. C. Galbraith, Uberreicht vom Verfasser Nicht einzeln im Buchhandel: Sonderdruck aus Zeitschrift fur Pflanzenphysiologie, Band 93, Heft 2, Seite 149-158 (1979) Gustav Fischer Verlag Stuttgart, West Germany. In the experiments on which that report is based, an attempt was made to mark freshly isolated protoplasts as well as suspension cultured cells but failed with respect to the freshly isolated protoplasts.